Effectorless fc molecules

ABSTRACT

The present invention provides molecules comprising a modified Fc region which do not mediate antibody effector functions such as antibody-dependent cellular cytotoxicity (ADCC) but maintain a long serum half-life due to binding to the neonetal Fc receptor (FcRn). To this end, the molecules of the present invention do not comprise the disulfide bridges of the antibody hinge region but are linked C-terminally by at least two covalent bonds. Furthermore, the molecules of the present invention comprise CH2 domains with an additional disulfide bond.

BACKGROUND

Monoclonal antibodies as well as Fc fusion proteins are very promisingbiopharmaceutical agents and the market for these compounds hasincreased significantly within the recent years. Reasons for the successof these molecules are their high specificity, safety and longhalf-lives in the circulation compared to small chemical compounds.Until 2017 more than 10 Fc fusion proteins have been approved and moreare under review. Antibodies are composed of two different proteinchains, a light chain (LC) and a heavy chain (HC). One LC is covalentlylinked to one HC and the HCs are on their parts covalently linked bydisulfide bonds. The LC is composed of a variable domain (VL) and aconstant domain (CL), whereas the HC consist of a variable domain (VH)and several constant domains (e.g., CH1, CH2 and CH3 domains for IgG).Functionally, an antibody can be divided into two Fab (Fragment antigenbinding) regions and one Fc (Fragment crystallizable) region. Each Fabregion consists of four domains: VL, CL, VH and CH1. The Fc regioncomprises the remaining constant domains (CH2 and CH3 domains for IgG).The Fab regions are connected to the Fc region via a flexible sequence,called the hinge region. This hinge region comprises disulfide bridgeswhich link the HCs.

Antibodies are multifunctional proteins. While antigens are bound by Fabregions, effector functions of antibodies are mediated by the Fc region.The effector functions of antibodies include antibody-dependent cellularcytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) andantibody-dependent complement deposition (ADCD). These effects aremediated via binding of the antibody Fc region to Fc gamma receptors(FcγRs) and the complement protein C1q, respectively. These antibodyeffector functions are important for the efficacy of natural antibodiesand also some engineered therapeutic antibodies, e.g., in oncology. Onthe other hand, the effector functions are undesired for many otherengineered therapeutic antibodies, e.g. antibodies against solubletargets such as pro-inflammatory cytokines. Such antibodies are oftendesigned to act mainly or only by blocking their specific targets viabinding of the Fab fragment, while any effector function of the Fcregion elicits an interfering and potentially harmful immune reaction,at the extreme a “cytokine storm” as observed for the anti-CD28 antibodyTGN1412.

On the other hand, the Fc region markedly prolongs half-life ofantibodies and Fc fusion proteins in the circulation via a “recycling”mechanism mediated by the neonatal Fc-receptor (FcRn). Moreover, the Fcregion contributes to the overall stability of the molecule and anotherimportant and desired function of the Fc-domain is its strong andspecific binding to protein A.

Binding of antibodies and Fc fusion proteins to immobilized protein A isusually the first purification step during the purification ofantibodies and Fc fusion proteins. Accordingly, a simple removal of theFc region from the antibody molecule would have severe drawbacks.

Therefore, several variants Fc regions have been developed to reduceantibody effector functions while maintaining the positive effects ofthe Fc region (half-life prolongation, protein A binding,stabilization). These approaches include the aglycosylated variants andvariants with point mutations (Wang et al., Protein Cell. 2018; 9(1):63-73). However, many of the point mutation variants retains atleast some antibody effector functions, while aglycosylated variantsexhibit reduced solubility or stability (Dumet et al., MAbs. 2019; 11(8): 1341-1350). Another attempt to abolish antibody effector functionsis the modification or deletion of the hinge region which contributes tothe binding of FcγRs and C1q to the Fc region (Vidarsson et al., FrontImmunol. 2014; 5:520). It was shown that such a “hinge-less” antibodydisplays virtually no FcγRs-mediated effector function (Valeich et al.,Antibodies (Basel). 2020 December; 9 (4): 50). However, these constructshad reduced stability and compromised binding to the FcRn which willtranslate in shorter in vivo half-life.

Thus, there remains the need for antibodies or Fc fusion proteins whichhave no antibody effector functions but maintain high stability,uncompromised FcRn binding and protein A binding. The present inventionaddresses this need by providing an Fc region which lacks a functionalhinge region but comprises at least two covalent bonds, which arelocated C-terminally to the Fc part, and an additional internaldisulfide bond in the CH2 domain.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a molecule comprisingtwo polypeptides,

-   -   wherein said polypeptides form an antibody Fc region,    -   wherein said two polypeptides are linked by at least two        covalent bonds which are located C-terminally to the portion of        each polypeptide which forms the Fc region,    -   wherein said two polypeptides are not linked by a disulfide bond        located N-terminally to the portion of each polypeptide which        forms the Fc region,    -   wherein each polypeptide comprises a CH2 domain which is part of        the Fc region, wherein each of these CH2 domains comprises an        additional internal disulfide bond which is not present in the        corresponding wild-type CH2 domain,    -   wherein the additional internal disulfide bond is selected from        the group consisting of disulfide bonds formed between cysteine        residues at the following positions according to EU numbering:        P238C and D265C; P238C and L328C; S267C and A327C; and V240C and        I332C.

In some embodiments, the additional internal disulfide bond is selectedfrom the group consisting of disulfide bonds formed between cysteineresidues at the following positions according to EU numbering: P238C andL328C; S267C and A327C; and V240C and I332C.

In some embodiments, the additional internal disulfide bond is selectedfrom the group consisting of disulfide bonds formed between cysteineresidues at the following positions according to EU numbering: P238C andL328C; and S267C and A327C.

In some embodiments, the two covalent bonds are two disulfide bonds.

In some embodiments, the at least two covalent bonds locatedC-terminally to the portion of each polypeptide which forms the Fcregion are comprised in a sequence corresponding to the amino acidsequence of an antibody hinge region.

In some embodiments, the affinity to C1q and/or FcγR1, FcγR2 and/orFcγR3 is reduced at least 10-fold, compared to the same moleculecomprising an antibody hinge region located N-terminally to the portionof each polypeptide which forms the Fc region.

In some embodiments, the affinity to protein A and/or FcgRn is notreduced, compared to the same molecule comprising an antibody hingeregion located N-terminally to the portion of each polypeptide whichforms the Fc region.

In some embodiments, the Fc region is the Fc region of IgG, IgM, IgA,IgD or IgE.

In some embodiments, the at least two covalent bonds are locatedC-terminally to a CH3 or CH4 domain on each polypeptide.

In some embodiments, the Fc region is the Fc region of an IgG antibody.

In some embodiments, the at least two covalent bonds are locatedC-terminally to a CH3 domain on each polypeptide.

In some embodiments, the molecule further comprises at least one activemoiety.

In some embodiments, the active moiety is located N-terminal of the CH2domain of at least one polypeptide and the molecule does not comprise anactive moiety which is located C-terminal of the at least two covalentbonds which are located C-terminally to the portion of each polypeptidewhich forms the Fc region.

In some embodiments, the active moiety is an antigen-binding moiety.

In a second aspect, the present invention provides a polynucleotideencoding a molecule according to any embodiment of the first aspect.

The present invention provides molecules which do not display thepotentially detrimental antibody effector functions (ADCC, ADCP, ADCD)of an Fc, but maintain all desired Fc functionalities: a high serumhalf-life via FcRn binding, easy purification via protein A binding andhigh molecule stability.

These advantageous properties of the molecules of the present inventionare due to the combination of the following features: a lack of afunctional antibody hinge region, which diminishes antibody effectorfunctions, and the at least two C-terminal covalent bonds, as well as anadditional internal disulfide bond in the CH2 domain, which bothenhances stability. Thus, the present invention provides molecules withno antibody effector functions, uncompromised FcRn binding and enhancedstability.

Compared to known Fc variants with point mutations which diminishantibody effector functions (e.g. the “LALA” mutation (L234A and L235A),the lack of an N-terminal hinge region, as in the molecules of thepresent invention, abolishes ADCD more effectively (FIG. 8 ).

While hinge-less antibodies without C-terminal covalent bonds have areduced stability and compromised FcRn binding (as shown in Valeich etal., Antibodies (Basel). 2020 December; 9 (4): 50), the molecules of thepresent invention are not compromised in FcRn binding.

In addition, a combination of the C-terminal covalent bonds and theadditional internal disulfide bond in the CH2 domains of molecules ofthe present invention stabilize the molecule and can thus fullycompensate for the lack of an N-terminal hinge region (Table 6).

The CH2 domains of the molecule comprise an additional internaldisulfide bond which is not present in the corresponding wild-type CH2domain. This further enhances the stability of the molecule. Certaininternal disulfide bonds provide a better stabilization than others. Interms of stabilization, particularly favorable internal disulfide bondsare P238C/L328C, S267C/A327C and V240C/I332C.

Moreover, the two polypeptides of the molecules of the present inventionform stable dimers due to the C-terminal at least two inter-chaincovalent bonds. The molecules adopt proper folding, no aggregationmediated by the additional C-terminal covalent bonds was observed (seeSEC-MALS data, FIGS. 4 and 11 ). Preventing aggregate formation is veryimportant for molecules which comprise an Fc region because aggregatescan mediate ADCD. Moreover, the C-terminal covalent bonds of moleculesof the present invention mask the natural Fc C-terminus of the antibodywhich also prevents ADCD mediated by Fc Terminus aggregation.

Furthermore, the molecules have a high expression level in standardprotein expression systems.

In some embodiments, the at least two C-terminal covalent bonds arecomprised in an amino acid sequence which corresponds to the amino acidsequence of an antibody hinge region. In this case, the at least twoC-terminal covalent bonds are at least two disulfide bonds within thissequence. Surprisingly, the sequence of the antibody hinge region formsinter-chain disulfide bonds also if positioned C-terminally to the Fcpart. As the sequence of the antibody hinge region can be derived from ahuman endogenous antibody hinge region, the usage of exogeneoussequences can be avoided which reduces the risk of anti-drug antibody(ADA) formation.

In some embodiments, the molecule comprises an active moiety locatedN-terminally of the Fc part. In some embodiments, the molecule does notcomprise an active moiety located C-terminally of the at least twocovalent bonds which are located C-terminally to the Fc region. This hasthe advantage that the C-terminal part of the Fc cannot interfere withthe active moiety, for instance does not hinder its binding to a target.In some embodiments, the active moiety is an antigen binding moiety.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 . Schematic view of an IgG with indicated binding sites forFc-binding proteins (A). FcγR and C1q are binding to the lower hingeregion (Vidarsson et al., Front Immunol. 2014; 5:520). This site isdistinct from the binding site of the FcRn and Protein A which islocated at the lower CH2 and upper CH3 domain (Vidarsson et al., FrontImmunol. 2014; 5:520). (B) Schematic view of an effector-less Fc regionaccording to the present invention. The hinge region, locatedN-terminally of the Fc region, was removed, its sequence was shifted tothe C-terminus of the Fc region. Moreover, the CH1 domain was connectedto the CH2 domains via a linker sequence. Consequently, this newarrangement completely eliminates the binding sites for FcγRs and C1q.

FIG. 2 . Overview of the different anti-TNFα antibody variants. The Fabis derived from adalimumab and is the same in all variants. Mab01 isadalimumab with IgG1 wild-type (wt). Mab02 is an IgG1 variant that hasthe known “LALA” (L234A/L235A) mutations as well as the N297A mutationto avoid glycosylation (Wang et al., Protein Cell. 2018; 9 (1):63-73).Mab03 has the IgG2 Fc-backbone which has a reduced effector functionscompared to IgG1. Mab04 has an IgG1 Fc-backbone with the known E269R andK322A mutations. Mab05 has an IgG1 Fc-backbone with the LALA(L234A/L235A) mutation and additional the known P329A mutation. Mab06 isan Fc IgG1 according to the present invention (labeled as “upside-down”)with the sequence of the hinge region transferred to the C-terminus andno linker sequence between CH1 and CH2 domains. Mab07 is an Fc IgG1according to the present invention (labeled as “upside-down”) furtherincluding a 19GS-linker between CH1 and CH2 domains.

FIG. 3 . SDS-PAGE of purified antibody variants according to FIG. 2under non-reducing and reducing conditions. M: Protein marker.

FIG. 4 . Analytical size exclusion chromatography and calculatedmolecular masses of the different antibodies. (A) The relative signalintensities of the Rayleigh ratio, as a measure of the scattered lightintensity, are plotted versus the elapsed time. (B) Close up view of thepeaks. The calculated molecular masses of the proteins within the mainpeak-fraction are indicated. Mab06 is labeled “ud-IgG1”; Mab07 islabeled “ud-IgG1 (19GS).

FIG. 5 . Binding of TNFα to the different antibodies as determined bySPR. Antibodies were immobilized on the chip and human TNFα was used asanalyte. The corresponding antibody variants are indicated. Mab06 islabeled “ud-IgG1”; Mab07 is labeled “ud-IgG1 (19GS).

FIG. 6 . SPR-affinity measurements of the different antibodies todifferent FcRs. For interaction analysis the FcRs were immobilized onthe sensor chip. The sensograms show the binding profiles of thedifferent antibodies at different concentrations. Mab06 is labeled“ud-IgG1”; Mab07 is labeled “ud-IgG1 (19GS).

FIG. 7 . Effector function: ADCC. (A) Schematic drawing of the assayprinciple of the assay. Upon binding of a membrane-standing antigen aneffector cell is activated upon FcγRIII activation. Activation of theeffector cell leads to cell lysis of the target cell. ADCC assay resultsobtained with (B) IgG1 wt, IgG1 LALA N297A (aglycosylated), and IgG1LALA P329A variant. (C) IgG1 wt, IgG1 E269R, K322A, and IgG2. (D) IgG1wt, Fc IgG1 according to the present invention (“ud-IgG1”), Fc IgG1according to the present invention including a 19GS-linker (“ud-IgG1(19GS)”). Activities are given in % specific lysis (see “Methods” in theExamples section).

FIG. 8 . Effector function: ADCD. (A) Schematic drawing of the assayprinciple of the assay. Formation of antigen-antibody complexes (immunecomplex) leads to recruitment of the complement system. The result isthe assembly of the membrane attack complex which kills the target cell.(B) IgG1 wt, IgG1 LALA N297A (aglycosylated), and IgG1 LALA P329Avariant. (C) IgG1 wt, IgG1 E269R, K322A, and IgG2. (D) IgG1 wt, Fc IgG1according to the present invention (“ud-IgG1”), Fc IgG1 according to thepresent invention including a 19GS-linker (“ud-IgG1 (19GS)”). Activitiesare given in % specific lysis (see Materials & Methods).

FIG. 9 . Overview of the different GLP1-RA-Fc fusion proteins. Allfusion proteins contain the same GLP1-sequence and linker sequence to anFc-domain. Note that GLP1-Fc01 and GLP1-Fc04 contain an IgG4 derivedFc-domain. This Fc variant contains the known F234A and L235A mutationsfor reduced effector function and the known S228P mutation to avoidhalf-antibody formation. All IgG1-Fc variants contain the LALA(L234A/L235A) mutations for reduced effector function. For sake ofclarity, these mutations are not depicted in the figure. Fc variantsaccording to the present invention (GLP-Fc04-GLP-Fc014 are labeled as“upside-down”.

FIG. 10 . SDS-PAGE of purified GLP1-RA-Fc fusion proteins variantsaccording to FIG. 9 under non-reducing (A) and reducing conditions (B).M: Protein marker.

FIG. 11 . Analytical size exclusion chromatography and calculatedmolecular masses of the different GLP1-RA Fc fusion proteins. Forclarity, only seven data sets are shown in one graph. (A, B) Therelative signal intensities of the Rayleigh ratio are plotted versus theelapsed time. (C, D) Close up view of the peaks. The calculatedmolecular masses of the proteins within the main peak-fraction areindicated.

FIG. 12 . Stability of GLP1-RA Fc fusion proteins as determined withnanoDSF. Maximum four variants are shown in one graph for clarity. (A)Variants GLP1-Fc01-GLP1-Fc04, (B) GLP1-Fc05-GLP1-Fc08, (C)GLP1-Fc09-GLP1-Fc12, and (D) GLP1-Fc13 and GLP1-14. All measurementswere done in duplicates. Curve represents mean data and the standarddeviation is indicated as shadow. The fluorescence intensity in eachsample is measured at 330 nm and 350 nm. In each upper panel the ratioof the fluorescence intensity at 350 nm/330 nm is plotted versus thetemperature. The 350 nm/330 nm ratio is a measure for a spectral shiftof tryptophan-residues induced upon unfolding of the protein. In thelower panels the first derivatives of the melting curves are shown. Themaxima of the first derivative were used to calculate the inflectionpoints (IP) as a measure of protein stability.

FIG. 13 . Pharmacodynamic profile of GLP1-Fc01 and GLP1-Fc04. Femaledb/db mice or lean controls were treated every fourth day s.c. for 14days with 10 nmol/kg GLP1-Fc01 or GLP1-Fc03 vs. vehicle (PBS) orcontrol. Body weight development (A) and fed blood glucose levels (B)was analyzed throughout the study, shown as glucose change from baseline(day 1). Timepoints of s.c. compound administration is indicated byarrows. Data are means+SEM, n=8 per group.

DETAILED DESCRIPTION

The present invention provides molecules comprising two polypeptides,wherein said polypeptides form an antibody Fc region, wherein said twopolypeptides are linked by at least two covalent bonds which are locatedC-terminally to the portion of each polypeptide which forms the Fcregion, wherein said two polypeptides are not linked by a disulfide bondlocated N-terminally to the portion of each polypeptide which forms theFc region, wherein each polypeptide comprises a CH2 domain which is partof the Fc region, wherein each of these CH2 domains comprises anadditional internal disulfide bond which is not present in thecorresponding wild-type CH2 domain, wherein the additional internaldisulfide bond is selected from the group consisting of disulfide bondsformed between cysteine residues at the following positions according toEU numbering: P238C and D265C; P238C and L328C; S267C and A327C; andV240C and I332C.

The molecule design of the present invention enables molecules which donot elicit the Fc-mediated antibody effector functions (ADCC, ADCP,ADCD), but maintain the desired function of the Fc region: FcRn binding,protein A binding and stabilization. The invention is based on thediscovery that the binding site for FcγRs and C1q, which are responsiblefor the antibody effector functions, are partially located in theantibody hinge region while the binding site for FcRn and protein A arelocated in the more C-terminal part of the Fc region (FIG. 1 ). Thus,removal of the hinge region abolishes antibody effector functionswithout impairing the desired Fc functions. Moreover, two C-terminalcovalent bonds and an additional internal disulfide bond in the CH2domain of the molecules of the invention overcome the limitations(compromised FcRn binding, reduced stability) observed for a plainhinge-less antibody variant (Valeich et al., Antibodies (Basel). 2020December; 9 (4): 50).

Definitions

Before the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodology, protocols and reagents described herein as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention, which will be limited onlyby the appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety.

As used in this description and the appended claims, the singular forms“a”, “an”, and “the” include plural referents, unless the contentclearly dictates otherwise.

Antibody positions: The position of amino acids within antibodymolecules or fragments of antibody molecules are specified according toEU numbering, unless indicated otherwise.

An “molecule”, as used herein, is group of atoms which are held togethereither by covalent interactions and/or by non-covalent interactions(e.g. hydrophobic or electrostatic interactions). In one embodiment, allatoms of the molecule are connected by covalent interactions.

In some embodiments, the molecule is an antibody. In some embodiments,the molecule is a multispecific antibody. In some embodiments, themolecule is an Fc fusion protein. In some embodiments, the molecule ofthe is a Fc fusion protein which comprises an Fc region and an activemoiety.

A “polypeptide”, as used herein, is a chain of 10 or more amino acidswhich are linked covalently by peptide bonds. Accordingly, the term“polypeptide” can stand for a chain of a multi-chain protein,independent of the length of such chain. In some embodiments, apolypeptide is a chain of a multi-chain protein.

An “Fc region”, as used herein, is a fragment of an immunoglobulinmolecule which is formed by several constant heavy chain (CH)immunoglobulin domains of two polypeptides. In natural antibodies, theFc region mediates binding to Fc receptors and components of thecomplement system. For natural IgG, IgA and IgD, the Fc region is formedby the CH2 and CH3 domains of both heavy chains. For natural IgM andIgE, the Fc region is formed by the CH2, CH3 and CH4 domains of bothheavy chains. The Fc regions of the present invention can be formed bythe same CH domains as the Fc region of natural antibodies, i.e. CH2 andCH3 domains or CH2, CH3 and CH4 domains. The Fc regions of the presentinvention comprise CH domains which are either identical to CH domainsof the natural immunoglobulins or are derived from CH domains of thenatural immunoglobulins, e.g. by inserting one or several pointsmutations.

“hinge region”, as used herein, is a part of an antibody sequence whichis located between the Fc region and the Fab regions. It providessegmental flexibility and can stabilize the antibody molecules. Thehinge region can be clearly defined based on structural data, e.g. theIgG1 hinge region comprises the residues 221-237 (R. Nezlin, “TheImmunoglobulins”, Academic Press, 1998, pages 23-26). In IgG antibodies,the hinge region comprises disulfide bonds which link the two heavychains of the antibody. IgG1 and IgG4 comprise 2 such disulfide bonds,while IgG2 comprises 4 and IgG3 comprises 11 (Liu and May, 2012 MAbs.2012 January-February; 4 (1): 17-23).

In the molecules of the present invention, a region is only designatedas “hinge region” if it is located N-terminally of the Fc region, as itis the case for all natural antibodies. If an amino acid sequence whichis identical or highly similar to a hinge region is placed C-terminallyof the Fc region (as in some embodiments of molecules of the presentinvention), it is designated as “sequence corresponding to the aminoacid sequence of an (antibody) hinge region”. Highly similar means atleast 70% identity to the sequence of an antibody hinge region and/orcomprising at least 7 contiguous amino acids of the sequence of anantibody hinge region.

A “covalent bond”, as used herein, is a chemical bond that involves thesharing of electron pairs between atoms. A covalent bond is distinctfrom non-covalent interaction, such as electrostatic interactions orhydrophobic effect.

The indication that a covalent bond, e.g. disulfide bonds, is “locatedC-terminally” of an Fc region means that, on both polypeptides formingsaid Fc region, the amino acid residues, e.g. cysteine residues, whichare involved in the formation of said covalent bond, are locatedC-terminally of the portion of the polypeptides which form the Fcregion.

The indication that no covalent bond, e.g. disulfide bond, is “locatedN-terminally” of an Fc region means that, on both polypeptides formingsaid Fc region, there are no amino acid residues, e.g. cysteineresidues, which are involved in the formation of such a covalent bond,located N-terminally of the portion of the polypeptide which form the Fcregion.

An “internal disulfide bond”, as used herein, is a disulfide bond formedby two cysteine residues within one immunoglobulin domain, e.g. a CHdomain. This means that an internal disulfide bond is always anintra-chain disulfide bond, not an inter-chain disulfide bond whichlinks different polypeptide chains. An “internal disulfide bond which isnot present in the corresponding wild-type CH2 domain”, also called“additional internal disulfide bond” is an internal disulfide bond whichis not present in the natural CH2 domain, i.e. the natural IgG, IgM,IgA, IgD or IgE CH2 domain, from which the modified CH2 of the inventionwas derived.

In some embodiments, the internal disulfide is selected from disulfidebonds formed between cysteine residues at the following positions: P238Cand D265C; P238C and L328C; S267C and A327C; and V240C and I332C.

A “linker”, as used herein, is a short and flexible amino acid sequencewhich links two regions of a molecule. For instance, a linker can link aFab region and the Fc region; or an active moiety and the Fc region; orthe Fc region and the C-terminal region of the molecule which comprisesthe at least two inter-chain covalent bonds. A typical linker consistsof 1-20 amino acids.

In some embodiments, the linker comprises several glycine and/or serineresidues. In some embodiments, the linker consists of glycine and/orserine residues. In some embodiments, the linker comprises an amino acidsequence which correspond to an amino acid sequence of a naturalantibody molecule. In some embodiments, the linker comprises an aminoacid sequence which correspond to an amino acid sequence of the naturalantibody molecule from which the CH2 and CH3 domains of the Fc regionare derived.

In one embodiment, the Fc region and the C-terminal region whichcomprises the at least two inter-chain covalent bonds are linked by alinker which consists of 6 amino acids. In one embodiment, the Fc regionand the C-terminal region which comprises the at least two inter-chaincovalent bonds are linked by a linker which consists of 1 amino acid.

An “active moiety”, as used herein, is a molecule, or part of amolecule, which exerts a biological function by interacting with abiological structure (e.g., a receptor for said molecule).

In some embodiments, the active moiety is a peptide. In someembodiments, the active moiety is a receptor agonist or receptorantagonist. In some embodiments, the active moiety is an enzyme. In someembodiments, the active moiety is a cytokine. In some embodiments, theactive moiety is an antigen-binding moiety.

An “antigen-binding moiety”, as used herein, is an antibody or antibodyfragment which is capable of binding to the target of such antibody.Examples of antigen-binding moieties are a Fab fragment, a F(ab)₂fragment, an scFv (single-chain variable fragment), an sdAb(single-domain antibody), a VHH, an ISVD (immunoglobulin single variabledomain), (e.g., a NANOBODY molecule), and a VNAR (variable new antigenreceptor).

Fc Region

In some embodiments of the present invention, the two polypeptides forman Fc region of an IgG, IgM, IgA, IgD or IgE antibody. In someembodiments of the present invention, the two polypeptides form an Fcregion of an IgG antibody. In some embodiments of the present invention,the two polypeptides form an Fc region of an IgG1 antibody. In someembodiments of the present invention, the two polypeptides form an Fcregion of an IgG2 antibody. In some embodiments of the presentinvention, the two polypeptides form an Fc region of an IgG3 antibody.In some embodiments of the present invention, the two polypeptides forman Fc region of an IgG4 antibody. In some embodiments of the presentinvention, the two polypeptides form a mixed Fc region which comprisesCH domains of different antibody classes or subclasses, e.g., IgG1 CH3domains and IgG4 CH2 domains.

C-Terminal Covalent Bonds

In some embodiments, the at least two covalent bonds are at least twodisulfide bonds. In some embodiments, the at least two covalent bondsare two disulfide bonds.

In some embodiments, the at least two covalent bonds are comprised in asequence corresponding to the amino acid sequence of an antibody hingeregion. In some embodiments, the sequence corresponding to the aminoacid sequence of an antibody hinge region is identical to the sequenceof an antibody hinge region. In some embodiments, the sequencecorresponding to the amino acid sequence of an antibody hinge region ishighly similar to the sequence of an antibody hinge region. In someembodiments, the sequence has at least 70% identity to the sequence ofan antibody hinge region. In some embodiments, the sequence has at least75% identity to the sequence of an antibody hinge region. In someembodiments, the sequence has at least 80% identity to the sequence ofan antibody hinge region. In some embodiments, the sequence has at least90% identity to the sequence of an antibody hinge region. In someembodiments, the sequence comprises at least 7 contiguous amino acids ofthe sequence of an antibody hinge region. In some embodiments, thesequence comprises at least 10 contiguous amino acids of the sequence ofan antibody hinge region. In some embodiments, the sequence comprises atleast 15 contiguous amino acids of the sequence of an antibody hingeregion.

In some embodiments, the sequence corresponding to the amino acidsequence of an antibody hinge region is identical or highly similar tothe sequence of an IgG4 hinge region. In some embodiments, the sequencecorresponding to the amino acid sequence of an antibody hinge region isselected from, ESKYGPPCPPCP (SEQ ID NO:1), ESKYGPPCPPCPA (SEQ ID NO:2),ESKYGPPCPPCPAPEAA (SEQ ID NO:3), GGGGSAESKYGPPCPPCP (SEQ ID NO:4),GGGGSAESKYGPPCPPCPA (SEQ ID NO:5) GGGGSAESKYGPPCPPCPAPEAA (SEQ ID NO:6).In some embodiments, the at least two covalent bonds are comprised in aproline-cysteine rich sequence. In some embodiments, saidproline-cysteine rich sequence is selected from GPPCPPCP (SEQ ID NO:7),GPPCPPCPA (SEQ ID NO:8), GPPCPPCPAPEAA (SEQ ID NO:9).

In some embodiments, the at least two covalent bonds are covalent bondscreated by click chemistry between non-natural amino acids.

In some embodiments, at least two covalent bonds are locatedC-terminally to a CH2, CH3 and/or CH4 domain on each polypeptide

In some embodiments, at least two covalent bonds are locatedC-terminally to a CH2 and/or CH3 domain on each polypeptide.

In some embodiments, at least two covalent bonds are locatedC-terminally to a CH2 and CH3 domain on each polypeptide.

Internal Disulfide Bond in CH2 Domain

Each polypeptide comprises a CH2 domain which is part of the Fc region,wherein each of these CH2 domains comprises an internal disulfide bondwhich is not present in the corresponding wild-type CH2 domain. Saidinternal disulfide bond is selected from the group consisting ofdisulfide bonds formed between cysteine residues at the followingpositions: P238C and D265C; P238C and L328C; S267C and A327C; and V240Cand I332C. In some embodiments, said internal disulfide bond is selectedfrom the group consisting of disulfide bonds formed between cysteineresidues at the following positions: P238C and L328C; S267C and A327C;and V240C and I332C. In some embodiments, said internal disulfide bondis selected from the group consisting of disulfide bonds formed betweencysteine residues at the following positions: P238C and L328C; and S267Cand A327C. In some embodiments, said internal disulfide bond is adisulfide bond formed between cysteine residues P238C and D265C. In someembodiments, said internal disulfide bond is a disulfide bond formedbetween cysteine residues P238C and L328C. In some embodiments, saidinternal disulfide bond is a disulfide bond formed between cysteineresidues S267C and A327C. In some embodiments, said internal disulfidebond is a disulfide bond formed between cysteine residues V240C andI332C. Disclosed is also an internal disulfide bond which is a disulfidebond formed between cysteine residues P238C and A327C.

In some embodiments, each of the polypeptide comprises a linker, whereinthe linker links the C-terminus of an immunoglobulin domain forming theFc region to one of the disulfide bonds located C-terminally of theimmunoglobulin domains. In some embodiments, said linker comprises 6amino acids.

Exemplary Embodiments

In some embodiments, the two polypeptides form an Fc region of an IgGantibody and the at least two covalent bonds are at least two disulfidebonds. In some embodiments, the two polypeptides form an Fc region of anIgG antibody and the at least two covalent bonds are two disulfidebonds. In some embodiments, the two polypeptides form an Fc region of anIgG antibody and the at least two covalent bonds are comprised in asequence corresponding to the amino acid sequence of an antibody hingeregion. In some embodiments, the two polypeptides form an Fc region ofan IgG antibody and the at least two covalent bonds are comprised in aproline-cysteine rich sequence. In some embodiments, the twopolypeptides form an Fc region of an IgG antibody and the at least twocovalent bonds are covalent bonds created by click chemistry betweennon-natural amino acids.

In some embodiments, the two polypeptides form an Fc region of an IgG1antibody and the at least two covalent bonds are at least two disulfidebonds. In some embodiments, the two polypeptides form an Fc region of anIgG1 antibody and the at least two covalent bonds are two disulfidebonds. In some embodiments, the two polypeptides form an Fc region of anIgG1 antibody and the at least two covalent bonds are comprised in asequence corresponding to the amino acid sequence of an antibody hingeregion. In some embodiments, the two polypeptides form an Fc region ofan IgG1 antibody and the at least two covalent bonds are comprised in aproline-cysteine rich sequence. In some embodiments, the twopolypeptides form an Fc region of an IgG1 antibody and the at least twocovalent bonds are covalent bonds created by click chemistry betweennon-natural amino acids.

In some embodiments, the two polypeptides form an Fc region of an IgG4antibody and the at least two covalent bonds are at least two disulfidebonds. In some embodiments, the two polypeptides form an Fc region of anIgG4 antibody and the at least two covalent bonds are two disulfidebonds. In some embodiments, the two polypeptides form an Fc region of anIgG4 antibody and the at least two covalent bonds are comprised in asequence corresponding to the amino acid sequence of an antibody hingeregion. In some embodiments, the two polypeptides form an Fc region ofan IgG4 antibody and the at least two covalent bonds are comprised in aproline-cysteine rich sequence. In some embodiments, the twopolypeptides form an Fc region of an IgG4 antibody and the at least twocovalent bonds are covalent bonds created by click chemistry betweennon-natural amino acids.

In some embodiments, the two polypeptides form an Fc region of an IgG1antibody and each of the CH2 domains of the Fc region comprises aninternal disulfide bond which is not present in the correspondingwild-type CH2 domain. In some embodiments, the two polypeptides form anFc region of an IgG1 antibody and each of the CH2 domains of the Fcregion comprises an internal disulfide bond which is not present in thecorresponding wild-type CH2 domain, wherein said internal disulfide bondis selected from the group consisting of disulfide bonds formed betweencysteine residues at the following positions: P238C and D265C; P238C andL328C; S267C and A327C; and V240C and I332C. In some embodiments, thetwo polypeptides form an Fc region of an IgG1 antibody and each of theCH2 domains of the Fc region comprises an internal disulfide bond whichis not present in the corresponding wild-type CH2 domain, wherein saidinternal disulfide bond is selected from the group consisting ofdisulfide bonds formed between cysteine residues at the followingpositions: P238C and L328C; S267C and A327C; and V240C and I332C.

In some embodiments, the two polypeptides form an Fc region of an IgG1antibody, the at least two covalent bonds are at least two disulfidebonds, and each of the CH2 domains of the Fc region comprises aninternal disulfide bond which is not present in the correspondingwild-type CH2 domain. In some embodiments, the two polypeptides form anFc region of an IgG1 antibody, the at least two covalent bonds are twodisulfide bonds, and each of the CH2 domains of the Fc region comprisesan internal disulfide bond which is not present in the correspondingwild-type CH2 domain. In some embodiments, the two polypeptides form anFc region of an IgG1 antibody, the at least two covalent bonds arecomprised in a sequence corresponding to the amino acid sequence of anantibody hinge region, and each of the CH2 domains of the Fc regioncomprises an internal disulfide bond which is not present in thecorresponding wild-type CH2 domain. In some embodiments, the twopolypeptides form an Fc region of an IgG1 antibody, the at least twocovalent bonds are comprised in a proline-cysteine rich sequence, andeach of the CH2 domains of the Fc region comprises an internal disulfidebond which is not present in the corresponding wild-type CH2 domain. Insome embodiments, the two polypeptides form an Fc region of an IgG1antibody, the at least two covalent bonds are covalent bonds created byclick chemistry between non-natural amino acids, and each of the CH2domains of the Fc region comprises an internal disulfide bond which isnot present in the corresponding wild-type CH2 domain.

In some embodiments, the two polypeptides form an Fc region of an IgG1antibody, the at least two covalent bonds are at least two disulfidebonds, and each of the CH2 domains of the Fc region comprises aninternal disulfide bond which is not present in the correspondingwild-type CH2 domain, wherein said internal disulfide bond is selectedfrom the group consisting of disulfide bonds formed between cysteineresidues at the following positions: P238C and L328C; S267C and A327C;and V240C and I332C. In some embodiments, the two polypeptides form anFc region of an IgG1 antibody, the at least two covalent bonds are twodisulfide bonds, and each of the CH2 domains of the Fc region comprisesan internal disulfide bond which is not present in the correspondingwild-type CH2 domain, wherein said internal disulfide bond is selectedfrom the group consisting of disulfide bonds formed between cysteineresidues at the following positions: P238C and L328C; S267C and A327C;and V240C and I332C. In some embodiments, the two polypeptides form anFc region of an IgG1 antibody, the at least two covalent bonds arecomprised in a sequence corresponding to the amino acid sequence of anantibody hinge region, and each of the CH2 domains of the Fc regioncomprises an internal disulfide bond which is not present in thecorresponding wild-type CH2 domain, wherein said internal disulfide bondis selected from the group consisting of disulfide bonds formed betweencysteine residues at the following positions: P238C and L328C; S267C andA327C; and V240C and I332C. In some embodiments, the two polypeptidesform an Fc region of an IgG1 antibody, the at least two covalent bondsare comprised in a proline-cysteine rich sequence, and each of the CH2domains of the Fc region comprises an internal disulfide bond which isnot present in the corresponding wild-type CH2 domain, wherein saidinternal disulfide bond is selected from the group consisting ofdisulfide bonds formed between cysteine residues at the followingpositions: P238C and L328C; S267C and A327C; and V240C and I332C. Insome embodiments, the two polypeptides form an Fc region of an IgG1antibody, the at least two covalent bonds are covalent bonds created byclick chemistry between non-natural amino acids, and each of the CH2domains of the Fc region comprises an internal disulfide bond which isnot present in the corresponding wild-type CH2 domain, wherein saidinternal disulfide bond is selected from the group consisting ofdisulfide bonds formed between cysteine residues at the followingpositions: P238C and L328C; S267C and A327C; and V240C and I332C.

In some embodiments, the molecule is an antibody and comprises onlyendogenous sequences. In some embodiments, the molecule is amultispecific antibody and comprises only endogenous sequences. In someembodiments, the molecule is a monospecific antibody and comprises onlyendogenous sequences.

In some embodiments, the molecule comprises an active moiety. In someembodiments, the active moiety is an antigen-binding moiety. In someembodiments, the active moiety is a peptide. In some embodiments, theactive moiety is a receptor agonist or receptor antagonist. In someembodiments, the active moiety is an enzyme. In some embodiments, theactive moiety is a cytokine. In some embodiments, the active moiety is achemokine. In some embodiments, the active moiety is a toxin. In someembodiments, the active moiety is a radiocontrast agent. In someembodiments, the active moiety is a nutrient.

In some embodiments, the molecule comprises an active moiety locatedN-terminally of the Fc part. In some embodiments, the molecule does notcomprise an active moiety located C-terminally of the at least twocovalent bonds which are located C-terminally to the Fc region. This hasthe advantage that the C-terminal part of the Fc cannot interfere withthe active moiety, for instance does not hinder its binding to a target.In some embodiments, the active moiety is an antigen binding moiety.

In some embodiments, each polypeptide comprises an antigen-bindingmoiety located N-terminally of the Fc part. In some embodiments, eachpolypeptide comprises the heavy chain of a Fab fragment locatedN-terminally of the Fc part. In some embodiments, each polypeptidecomprises an ISVD located N-terminally of the Fc part.

Polynucleotides, Vectors, Cell

In a second aspect, the present invention relates to one or morepolynucleotides encoding a molecule of the present invention. Thisrefers to all embodiments described above. The one or morepolynucleotides according to the second aspect may also encode the oneor more additional polypeptides comprised in a complex with the moleculeof the present invention. In one embodiment, the one or morepolynucleotides encode an antibody or antibody-like structure. In oneembodiment, the one or more polynucleotides are isolated.

In a third aspect, the present invention relates to one or moreexpression vectors comprising the one or more polynucleotides accordingto the second aspect of the invention. The term “vector” as used hereinrefers to any molecule (e.g., nucleic acid, plasmid, or virus) that isused to transfer coding information to a host cell. The term “vector”includes a nucleic acid molecule that is capable of transporting anothernucleic acid to which it has been fused. One type of vector is a“plasmid,” which refers to a circular double-stranded DNA molecule intowhich additional DNA segments may be inserted. Another type of vector isa viral vector, wherein additional DNA segments may be inserted into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) can be integrated intothe genome of a host cell upon introduction into the host cell andthereby are replicated along with the host genome. In addition, certainvectors are capable of directing the expression of genes they comprise.Such vectors are referred to herein as “expression vectors”.

In a fourth aspect, the present invention relates to a cell comprisingthe one or more polynucleotides according to the second aspect of theinvention or the one more expression vectors according to the thirdaspect of the invention. A wide variety of cell expression systems canbe used to express said polynucleotides including the use of prokaryoticand eukaryotic cells, such as bacterial cells (e.g. E. coli), yeastcells, insect cells or mammalian cells (e.g. mouse cells, rat cells,human cells etc.). For this purpose, a cell is transformed ortransfected with said polynucleotide(s) or expression vector(s) suchthat the polynucleotide(s) of the invention are expressed in the celland, in one embodiment, secreted into the medium in which the cells arecultured, from where the expression product can be recovered.

CH2 Domains With Internal Disulfide Bonds

In another aspect, the present invention provides a CH2 domain, whereinsaid CH2 domain comprises an internal disulfide bond which is notpresent in the corresponding wild-type CH2 domain. In some embodiments,the present invention provides an IgG1 CH2 domain, wherein said CH2domain comprises an internal disulfide bond which is not present in thewild-type IgG1 CH2 domain. In some embodiments, said internal disulfidebond in the IgG1 CH2 domain is selected from the group consisting ofdisulfide bonds formed between cysteine residues at the followingpositions according to EU numbering: P238C and D265C; P238C and A327C;P238C and L328C; S267C and A327C; and V240C and I332C. In someembodiments, said internal disulfide bond in the IgG1 CH2 domain isselected from the group consisting of disulfide bonds formed betweencysteine residues at the following positions: P238C and L328C; S267C andA327C; and V240C and I332C. In some embodiments, said internal disulfidebond in the IgG1 CH2 domain is a disulfide bond formed between cysteineresidues P238C and D265C. In some embodiments, said internal disulfidebond is a disulfide bond formed between cysteine residues P238C andA327C. In some embodiments, said internal disulfide bond in the IgG1 CH2domain is a disulfide bond formed between cysteine residues P238C andL328C. In some embodiments, said internal disulfide bond in the IgG1 CH2domain is a disulfide bond formed between cysteine residues S267C andA327C. In some embodiments, said internal disulfide bond is a disulfidebond formed between cysteine residues V240C and I332C.

In some embodiments, the present invention provides a CH2 domain,wherein said CH2 domains comprises at least two internal disulfide bondswhich are not present in the corresponding wild-type CH2 domain. In someembodiments, the present invention provides an IgG1 CH2 domain, whereinsaid CH2 domains comprises at least two internal disulfide bonds whichare not present in the wild-type IgG1 CH2 domain. In some embodiments,said internal disulfide bonds in the IgG1 CH2 domain are selected fromthe group consisting of disulfide bonds formed between cysteine residuesat the following positions according to EU numbering: P238C and D265C;P238C and A327C; P238C and L328C; S267C and A327C; and V240C and I332C.In some embodiments, said internal disulfide bonds in the IgG1 CH2domain are formed between cysteine residues at the following positions:P238C and D265C as well as S267C and A327C; P238C and D265C as well asV240C and I332C; P238C and A327C as well as V240C and I332C; P238C andL328C as well as S267C and A327C; P238C and L328C as well as V240C andI332C; S267C and A327C as well as V240C and I332C.

It is also possible to introduce additional disulfide bonds to stabilizethe molecule, e.g. intradomain disulfide bonds in the CH3 domain asdescribed in the art (Wozniak-Knopp PLOS One. 2012; 7 (1): e30083).

In some embodiments, the CH2 domain which comprises an internaldisulfide bond which is not present in the corresponding wild-type CH2domain is part of a larger molecule. In some embodiments, the moleculeis an antibody. In some embodiments, the molecule is a multispecificantibody. In some embodiments, the molecule is a monospecific antibody.

Examples

Example 1 shows deals with the anti-TNF antibody adalimumab, whileExample 2 employs a GLP1 receptor agonist (RA) Fc fusion protein.

Methods

The following section summarizes the methods which were used in theExamples below.

Protein Constructs and Protein Expression

All proteins were transiently expressed in CHO or HEK293 cells. Thesequences of the different antibody variants are shown in the sequencelisting. The DNAs for the different protein chains were synthesized(Thermo Fisher Scientific) and cloned into an expression vector under aCMV promotor sequence and a leader sequence required for proper proteinsecretion into the culture supernatant. Expression and purification weredone essentially as described by Becker et al. (2019) Protein ExprPurif. 2019 January; 153:1-6. In brief, cells were transfected using PEIin a ratio of DNA: PEI of 1:3. Expression was done for 6 days and cellswere separated from the culture supernatant by centrifugation (20 min,3000 g, 4° C.). Culture supernatants were 0.22 μm filtered and loadedonto A MabSelect Sure column (GE Healthcare) equilibrated in phosphatebuffered saline (PBS, Gibco/Thermo Fisher Scientific). After elution,proteins were desalted and further purified using a Superdex 200 (GEHealthcare) size exclusion chromatography column equilibrated with PBS(Gibco).

The sequences for the GLP1-RA Fc fusion proteins were either directlyflanked by a leader sequence or a leader sequence containing a His-tagfollowed by a Tev-cleavage site. For purification via the His-tag,proteins were captured on a His-tag purification resin (complete, Roche)column, equilibrated in 300 mM NaCl, 50 mM Tris, pH 8.0. Column waswashed with 300 mM NaCl, 50 mM Tris, pH 8.0, 5 mM imidazole and theprotein was eluted with 300 mM NaCl, 50 mM Tris, pH 8.0, 500 mMimidazole. Tev-cleavage was done at room temperature in 100 mM NaCl, 50mM, 50 mM Tris, pH 8.0 overnight and the cleavage mixture was directlyloaded on a protein A column and further purified. When a His-tag wasnot present, the proteins were purified using protein A as describedabove.

SDS-PAGE Analysis

Protein samples (5 μg protein) were mixed with either 4×LDS samplebuffer (Life Technologies/Thermo Fisher Scientific) or 4×LDS samplebuffer with 50 mM dithiothreitol for SDS-PAGE analysis undernon-reducing or reducing conditions, respectively. Samples wereincubated for 5 min at 99° C. before loading on a 4-12% SDS-PAGE withMES as running buffer (Life Technologies/Thermo Fisher Scientific).BenchMark protein ladder was used as marker (Invitrogen/Thermo FisherScientific).

Analytical Size Exclusion Chromatograph and Multi-Angel Static LightScattering Measurements (SEC-MALS)

Analytical size exclusion chromatography (SEC) runs were done using aSuperdex 200 10/HR column (GE Healthcare) equilibrated in freshlydegassed and 0.2 μm filtered PBS (Gibco). Sample volume was 50 μl forall samples and protein concentration was adjusted to 3 mg/ml for allsamples. An ÄKTA purifier (GE Healthcare) was used as chromatographysystem. The system was connected to a multi-angle static lightscattering (MALS) detector (miniDawn, Wyatt) and a refractive index(RI)-detector (Shodex RI-101, Showa-Denko) as concentration detector.Data analysis was done using the ASTA 5.3.4.20 (Wyatt) software. Arefractive index increment (dn/dc) value of 0.185 was used for allsamples.

MS-Analysis

Protein integrity was checked by LC-MS. Protein samples weredeglycosylated with 12.5 μg of protein diluted to 0.5 mg/ml in ddH₂Ocontaining PNGaseF (1:50 v/v) (glycerol free, New England Biolabs) at37° C. for 15 h. If samples were analysed under reduced conditions 0.1 Mdithiothreitol was added. The LC-MS analysis was done on an Agilent 6540Ultra High Definition (UHD) Q-TOF equipped with a dual ESI interface andan Agilent 1290/1260 Infinity LC System. Reversed phase (RP)chromatography was done using a PLRP-S 1000A 5 μm, 50×2.1 mm (Agilent)with a guard column PLRP-S 300A 5 μm, 3×5 mm (Agilent) at 200 μL/min and80° C. column temperature.

1 μg protein was injected onto the column and eluted with a lineargradient from 0 to 17 min with increasing acetonitrile concentration.Eluents used were buffer A containing LC water and 0.1 formic acid andbuffer B containing 90% acetonitrile, 10% LC water and 0.1 formic acid.Obtained mass data were analysed using the software MassHunterBioconfirm B.06 (Agilent). Molecular masses were calculated based on theamino acid sequences of the proteins using GPMAW software version 10.32(Lighthouse Data, Denmark).

Surface Plasmon Resonance Analysis

All analysis of antibody binding to FcRs were performed on a BiacoreT200 instrument (GE Healthcare). FcγR binding assays were done usinganti-His capture. Anti-tetra His (Qiagen) was buffer exchanged into PBSpH 7.2 (Gibco), diluted to 25 μg/mL in 10 mM sodium acetate pH 4.0 anddirectly immobilized to a series S CM5 chip to a surface density of˜10,000 RU using the amine coupling kit provided by GE Healthcare.His-tagged recombinant human and mouse FcγRs (FcγRI/CD64 #1257-FC;FcγRIIa/CD32a (R167) #1330-CD; FcγRIIb/c/CD32b/c #1875-CD;FcγRIIIa/CD16a #4325-FC; FcγR4/CD16-2 #4325-FC; R&D Systems) werediluted to 2 μg/mL in HBS-EP+ (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mMEDTA, 0.05% surfactant P20) and injected for 30 sec at 10 μL/minflowrate. Antibodies were serially diluted 3-fold from 3000 to 37 nM andinjected over the captured receptors for 2 min in duplicate. For bindingto hFcγRI, serial dilutions were made from 300 nM to 3.7 nM. The surfacewas regenerated with 10 mM glycine pH 1.5 for 30 sec. Sensorgrams wereprocessed using the BiaEvaluation software (GE Healthcare) and fit to a1:1 binding model to obtain kinetic constants. FcRn binding measurementswere done using biotinylated FcRn. Biotinylated human FcRn protein waspurchased from Immunitrack (#ITF01). A capture assay using a biotincapture kit (GE Healthcare) for reversible capture of the biotinylatedFcRn was used. The CAP chip surfaces were prepared by injecting thebiotin capture reagent solution for 300 sec at a flow rate of 2 μL/min.The FcRn protein was captured at a concentration of 0.5 μg/mL and a flowrate of 10 μL/min for 90 sec to reach a capture level of typically 100RU. The analyzed proteins were used in a 1:1 dilution series inHBS-EP+buffer from 1600 nM to 12.5 nM. The proteins were injected for 60sec and dissociation by buffer inject was measured for 120 sec. HBS-EP+buffer as used as assay buffer at a flow rate of 30 μl/min. Chipsurfaces were regenerated with the regeneration solution of the biotincapture kit as described by the manufacturer. Binding curves wereanalyzed in the BIAcore T200 evaluation software v2.0 using a flow cellwithout captured FcRn as reference and subtraction of a buffer inject(double referencing). The equilibrium dissociation constants (KD) weredetermined by global steady state data fitting of the equilibriumbinding responses (Req) for each protein concentration.

Binding of human TNFα to antibodies was performed using surface plasmonresonance on a Biacore 3000 instrument (GE Healthcare). The antibodieswere immobilized on the chip and TNFα was used as analyte. For captureof the antibodies the human antibody capture kit (GE Healthcare) wasused. The capture antibody was immobilized via primary amine groups(typically 10,000 RU) on a research grade CM5 chip (GE Life Sciences)using standard procedures as described for the human antibody capturekit. The analyzed antibody was captured at a flow rate of 10 μL/min withan adjusted RU value that would result in maximal analyte binding signalof typically 30 RU. Binding traces were measured against recombinanthuman TNFα (Sigma-Aldrich, #H8916) at a concentration of 100 nM withassociation and dissociation times of 240 sec and 300 sec, respectively.HBS-EP was used as assay buffer at a flow rate of 30 μl/min. Chipsurfaces were regenerated with the regeneration solution of the humanantibody capture kit. Binding traces were analyzed in the BIAevaluationprogram package v4.1 using a flow cell without captured antibody asreference and subtraction of a buffer inject (double referencing).

ADCC and ADCD In Vitro Assays

The target cells were generated by transfecting a CHO-K1 cell line witha mutant form of human TNFα. This mutant form is known to be only poorlycleaved by membrane proteases and remains at the cell surface. ThisTNFα-form was cloned into a mammalian expression vector under the CMVpromotor further containing a neomycine resistance gene for selection.Cells were grown in Ham's F12 medium (Biowest, Nuaillé, France)containing 10% heat-inactivated fetal bovine serum (FBS, Gibco) and 1mg/mL G418 antibioticum (Invivogen, Toulouse, France) for selection.Both assays use the same target cell and are based on the chromium(⁵¹Cr) release assay. For ADCD assays, target cells were washed with PBSand incubated with trypsin for 4 minutes. Cells were labelled with ⁵¹Cr,washed and plated in 96 well plates at approx. 3000 cells per well andincubated with RPM1-1640 (Biowest) medium containing 5% FBS. Guinea pigcomplement (Standard complement, Tebubio) at a fixed concentration andantibodies were added at different concentrations and further incubatedfor 4 hours at 37° C. For radioactivity measurements, aliqots werewithdrawn, transferred into a LumaPlate (PerkinElmer) and gamma countingwas done using a MicroBeta Jet device (PerkinElmer). ADCC assays wereperformed in a similar manner as described for the ADCD assay butinstead of adding complement, effector cells were added at a ratio of10:1 (effector:target cell). The used effector cell were monoclonalhuman cytotoxic T lymphocytes expressing the human FcγRIIIa (V158)receptor as described in the literature (Clémenceau et al., Blood. 2006;107 (12):4669-4677). In all assays, negative controls were included wereneither complement or effector cells, respectively, were added(spontaneous release). Maximum ⁵¹Cr release was achieved by adding 0.75%Triton X-100 to the cell culture medium. Results are expressed as %specific release and is calculated following the equation: % specificrelease=(experimental release−spontaneous release)/(maximumrelease−spontaneous release). All measurements were done in triplicates.

Pharmacodynamics In Mice

The mice studies were performed at Covance (Covance Laboratories Inc,Greenfield, IN). All procedures were in compliance with the U.S.Department of Agriculture's (USDA) Animal Welfare Act (9 CFR Parts 1, 2,and 3) and the Guide for the Care and Use of Laboratory animals(Institute for Laboratory Animal Research.

Female db/db (BKS.Cgo−+Leprdb/+Leprdb/OlaHsd) or lean mice(BKS.Cg−[lean]/OlaHsd) were from Envigo. Animals were 12 weeks old atstudy start and grouped with n=8 animals. Mice were feed with Purina5008 ad libitum, had free access to water and were maintained on a 12 hlight/dark cycle. As a stratification criterion HbA1c values (hemolysatemethod) were determined at day 7 of the predose phase. HbA1c is a is abiomarker for the average blood glucose level during the preceding ˜4weeks. Low, medium and high HbA1c values were spread as equally aspossible across groups resulting in similar Hb1Ac group-means. Allanimals were treated once every 4 days s.c. with an application volumeof 5 mL/kg with vehicle (PBS, Gibco Ref 14190) or compound at 10nmol/kg. Blood samples were withdrawn from the tail tip withoutanaesthesia and blood glucose was measured with a glucometer on day 9 ofthe predose-phase and days 1, 5, 9 and 13 (0 and 4 hr postdose) as wellas days 2, 3, 4, 6, 7, 8, 10, 11, 12, and 14 of the dosing phase. Bodyweights were measured daily.

GLP1-Receptor Activity Assays

Agonism of compounds for human glucagon-like peptide-1 (GLP1) receptorwas determined by functional assays measuring CAMP response ofrecombinant PSC-HEK-293 cell line stably expressing human GLP1 receptor.Cells were grown in a T175 culture flask placed at 37° C. to nearconfluence in medium (DMEM/10% FBS) and collected in 2 ml vials in cellculture medium containing 10% DMSO in concentration of 1-5×10⁷ cells/ml.Each vial contained 1.8 ml cells. The vials were slowly frozen to −80°C. in isopropanol, and then transferred in liquid nitrogen for storage.Prior to their use, frozen cells were thawed quickly at 37° C. andwashed (5 min at 900 rpm) with 20 ml cell buffer (1×HBSS; 20 mM HEPES,plus 0.1% HSA if indicated in example conditions). Cells wereresuspended in assay buffer (cell buffer plus 2 mM IBMX) and adjusted toa cell density of 1×106 cells/ml. For measurement, 5 μl cells (final5000 cells/well) and 5 μL of test compound were added to a 384-wellplate, followed by incubation for 30 minutes at room temperature. TheCAMP content of cells was determined using homogenous time resolvedfluorescence technique. (HTFR, CisBio). After addition of HTRF reagentsdiluted in lysis buffer (kit components), the plates were incubated for1 h, followed by measurement of the fluorescence ratio at 665/620 nm. Invitro potency of agonists was quantified by determining theconcentrations that caused 50% activation of maximal response (EC50).

Thermal Stability Measurements

Analysis of thermal stability was done using a Prometheus NT Flex device(NanoTemper Technologies) based on the nanoDSF technology. The device isequipped with Aggregation Optics allowing collection of scatteringinformation simultaneously with fluorescence measurements. Measurementswere done in the range from 20° C. to 95° C. with a thermal ramp of 1°C./min following the instructions of the manufacturer. All proteinsamples were dissolved in PBS and protein concentration was adjusted to0.5 mg/mL. Measurements were done in duplicates. Data analysis was doneusing the software PR ThermControl V.2.1 (NanoTemper Technologies).

Example 1: Generation of Fc Variants of Adalimumab According to thePresent Invention

The concept of Fc molecules according to the present invention wastested for the anti-TNF antibody adalimumab. We created two differentversions of an Fc adalimumab according to the present invention (FIG. 2, mab06 (heavy chain SEQ ID NO: 10) and mab07 (heavy chain SEQ ID NO:11)). These two constructs differed only in the length of the linkerreplacing the hinge region. For comparison purposes, Fc-backbonevariants known to have silenced effector functions were made as well(FIG. 2 , mab02, mab04, mab05). The Fab was the same in all constructs.All antibodies were transiently expressed in CHO or Hek293 cells andpurified using a two-step purification procedure. Interestingly, thehighest yields were obtained with the Fc variants according to thepresent invention (Tab. 1).

TABLE 1 Obtained yields (after two-step purification) for the differentantibody variants. Fc-backbone Yield [mg/L] IgG1 IgG2 100.4 udIgG1 200.7udIgG1 (19 GS linker) 150.6 IgG1 LALA N297A 120.7 IgG1 LALA P239A 90.4IgG1 E269R, K322A 140.7

All antibodies were analyzed for homogeneity by SDS-PAGE (FIG. 3 ) andanalytical size exclusion chromatography coupled with MALS. Lightscattering measurement is a very sensitive method for the detection oflarge particle, e.g. aggregates. Furthermore, it allows the calculationof molecular masses without the need for reference molecules. In allantibody samples the presence of aggregates was below the detectionlimit (FIG. 4 ). The obtained calculated molecular masses for allantibodies was in good accordance with the calculated values. Inaddition, identity of all antibody variants was verified by MS (Tab. 2).

TABLE 2 Calculated masses and masses obtained by SEC-MALS and MS foreach antibody. Molecular masses were calculated with GPMAW (average massvalues). All cysteines were assumed to form disulfide-bridges.Calculated Mw Mw by SEC- Mw by MS Antibody (Da) non-reduced MALS (Da)(intact) (Da) mab01 145,189 138,700 145,197 mab02 144,935 134,800144,940 mab03 144,743 137,300 144,751 mab04 145,129 136,200 145,138mab05 144,969 137,900 144,976 mab06 145,409 137,200 145,418 mab07147,785 136,800 147,793

The Fc IgG1 variant according to the present invention with 19GS-linker(mab07) elutes slightly earlier from the SEC column. In this molecule,the Fab domains are connected via long linker (GGGGGGGGGGGGSGGGGSA, SEQID NO: 12) to the Fc region. This arrangement obviously allows a moreopen molecule configuration resulting in a slightly enhancedhydrodynamic radius.

Binding to the antigen, human TNFα, was checked by surface plasmonresonance-measurements (FIG. 5 ).

Binding Assays to FcRs

The Fc variants of adalimumab according to the present invention lackthe FcγR binding site located in the IgG hinge region (FIG. 1 ). Thus,they should not bind to FcγRs. To check whether binding of the Fcvariants according to the present invention to the FcγRs was indeedabolished we measured binding to immobilized FcγRs using surface plasmonresonance in comparison to other antibody variants carrying silencingmutations. We analyzed binding to human FcγRI, FcγRIIa, b and c, andFcγRIIIa. Moreover, we included the murine activating FcγRIV since manyantibodies and Fc fusion proteins are analyzed in mouse models. Finally,the human FcRn receptor, which is important for recycling of antibodiesand Fc fusion proteins, was also included in the test panel. The resultsare summarized in Tab. 3 and the corresponding sensorgrams are shown inFIG. 6 .

TABLE 3 Affinity measurements of the different anti-TNFα-antibodies todifferent human FcγRs. Kds were determined, where possible, using a 1:1kinetic model (n/d: no binding detected; *too low: signal was too low toobtain binding constants). hFcγRI hFcγRIIa(R167) hFcγRIIb/chFcγRIIIa(V158) mFcγRIV hFcRn Antibody Kd [nM] Kd [nM] Kd [nM] Kd [nM]Kd [nM] Kd [nM] mab01 0.8 3280 >10000 656 827 361 mab02 n/d n/d n/d n/dn/d 263 mab03 *too low >5000 >10000 >10000 >10000 342 mab04 6.7 *too lown/d >10000 >5000 357 mab05 >10000 n/d n/d n/d n/d 265 mab06 n/d n/d n/dn/d n/d 528 mab07 *too low n/d n/d n/d n/d 348

The unmodified IgG1 backbone (mab01) binds to all FcγR. The highestbinding affinity was obtained with FcγRI, which is known as a highaffinity binding receptor. An unmodified IgG2 (mab03), tested forcomparison purposes, shows some binding to FcRγIIa, FcγRIIb/c and to alower extent to FcRγIIIa. The antibodies with the Fc IgG1 according tothe present invention (mab06, mab07) showed no detectable binding to alltested FcγRs. This is also true for the IgG1 LALA N297A (mab02) and IgG1LALA P329A (mab05) backbone variants. For the IgG1 E269R, K322A variant(mab04) a high affinity binding to FcγRI could still be measured.Binding of this variant to the FcγRIIIa and murine FcγRIV wassignificantly reduced but still detectable. All analyzed Fc variantsshow similar binding affinity for the hFcRn.

Functional Cellular Assays (ADCC and ADCD)

ADCC and ADCD are the most important mechanisms for the effectorfunctions of antibodies. Thus, we wanted to ensure that the Fc variantsof adalimumab according to the present invention do not elicit ADCC orADCD. In an ADCC assay the target cells expressing the membrane-standingantigen is incubated with the antibody of interest and effector cells.If an effector cell is tethered to the target cell it gets activated andkills the target cell (FIG. 7A). All analyzed antibody variants show nodetectable specific target cell lysis, while the IgG1 wt variantprovokes significant specific cell lysis (FIG. 7B-D).

The ADCD mechanism leads to cell death by activation of the complementsystem. The complement cascade is triggered by binding of the C1qprotein to antigen-antibody complexes on the cell surface of the targetcell. The final step of complement cascade activation is the formationof the so-called membrane attack complex which leads to cell lysis (FIG.8A). The IgG1 LALA N297A (mab02) and the IgG2 backbone (mab03) are stillable to induce significant specific cell lysis (FIG. 8B, C). The IgG1E269R, K322A variant (mab04) triggered a lower but still detectablespecific cell lysis. For the Fc IgG1 variants according to the presentinvention (mab06, mab07) and the IgG1 LALA P329A variants (mab05) no orno significant specific cell lysis was observed (FIG. 8B, D).

Examples 2: GLP1-Receptor-Agonist Fc-Domain Fusion Proteins

To evaluate the Fc format according to the present invention for apeptide Fc fusion protein and to test in vivo functionality, we usedGLP1-receptor agonist (RA) Fc fusion proteins. Thus, we fused a humanGLP1-RA via a peptide linker to an IgG4 Fc region (FIG. 9 , GLP1-Fc01and 04) or to an IgG1 Fc region FIG. 9 , GLP1-Fc02, 05-014). FIG. 9shows an overview of the different GLP1-RA-Fc fusion protein variants.

In addition to simple Fc variants according to the present invention forIgG4 (GLP1-Fc04) and IgG1 (GLP1-Fc05), we created 9 Fc variantsaccording to the present invention for IgG1 (GLP1-Fc06-14) whichcomprise an additional internal disulfide bond in the CH2 domain. Thisis intended to additionally stabilize the CH2 domain since it is knownthat the IgG CH2 domain unfolds earlier than the IgG CH3 domain. While 6of the variants with an additional internal disulfide bond had asequence derived from the wild-type hinge region sequence at theC-terminus (GLP-Fc06-11), the other 3 variants had a shortenedproline-cysteine rich sequence at the C-terminus (GLP-Fc12-14). AllGLP1-Fc-RA fusion proteins were analyzed for homogeneity by SDS-PAGE(FIG. 10 ) and analytical size exclusion chromatography coupled withMALS (FIG. 11 ). In all analyzed samples, presences of aggregates wasbelow detection limit and the obtained molecular weight were in goodaccordance with the calculated molar mass of a homo-dimeric molecule(Tab. 4).

TABLE 4 Calculated masses and masses obtained by SEC-MALS and MS forGLP1-Fc fusion proteins. Molecular masses were calculated with GPMAW(average mass values). All cysteines were assumed to formdisulfide-bridges. Calculated Mw by Mw by MS Calculated Mw Mw SEC- non-Mw by MS non-reduced MALS reduced reduced reduced Protein (Da) (Da) (Da)(Da) (Da) GLP1-Fc01 59,670 62,690 59,675 29,841 29,840 GLP1-Fc02 60,38260,560 n.d. 30,193 30,194 GLP1-Fc03 59,608 61,580 n.d. 29,808 29,808GLP1-Fc04 63,770 64,480 n.d. 31,891 31,891 GLP1-Fc05 59,545 59,180 n.d.29,772 n.d. GLP1-Fc06 59,533 60.620 n.d. 29,766 n.d. GLP1-Fc07 59,62160,013 n.d. 29,810 n.d. GLP1-Fc08 59,537 59,750 n.d. 29,768 n.d.GLP1-Fc09 59,641 60,014 n.d. 29,820 n.d. GLP1-Fc10 59,533 60,790 n.d.29,766 n.d. GLP1-Fc11 58,914 60,150 n.d. 29,457 n.d. GLP1-Fc12 57,75759,400 n.d. 28,878 n.d. GLP1-Fc13 57,749 58,900 n.d. 28,874 28,871GLP1-Fc14 57,853 57,750 n.d. 28,926 28,925 N.d.: not determined

For comparison purposes, we included also a variant without any covalentbond between the heavy chains (GLP1-Fc03). In contrast to the othertested variants, this GLP1-Fc03 variant showed a clear dissociation inthe SDS-PAGE under non-reducing conditions.

The activity for all variants was checked in a GLP1-R activation assay.To ensure that the introduced disulfide-bonds do not interfere withbinding to the FcRn, binding to FcRn was checked using surface plasmonresonance technology at pH 6.0. GLP1 activity data and binding data toFcRn are shown in Tab. 5.

TABLE 5 Activity data and binding data to the FcRn. EC50 values weredetermined with a GLP1R expressing reporter cell line. Binding to thehuman FcRn was measured using surface plasmon resonance technique andthermal stability measurements were done with nanoDSF. EC50 EC50 hFcRnmFcRn hGLP1R mGLP1R (pH6.0) (pH6.0) Variant [pMol] [pMol] Kd [nM] Kd[nM] GLP1-Fc01 2.08 1.47 514 306 GLP1-Fc02 4.12 n.d. 637 n.d. GLP1-Fc031.69 0.84 315 146 GLP1-Fc04 2.21 0.99 500 233 GLP1-Fc05 2.59 n.d. 451n.d. GLP1-Fc06 102.8 n.d. 363 n.d. GLP1-Fc07 3.19 n.d. 519 n.d.GLP1-Fc08 4.16 n.d. 558 n.d. GLP1-Fc09 4.87 n.d. 868 n.d. GLP1-Fc10 2.75n.d. 862 n.d. GLP1-Fc11 2.95 n.d. 814 n.d. GLP1-Fc12 2.40 n.d. 497 n.d.GLP1-Fc13 0.48 n.d. n.d. n.d. GLP1-Fc14 0.18 n.d. n.d. n.d. GLP1 (ctrl)0.58 0.71 n.d. n.d. n.d.: not determined.

Thermal stability of GLP1-RA-Fc fusion proteins was analyzed with theDSF method (FIG. 12 , Tab. 6).

TABLE 6 Stability of GLP1-RA-Fc variants as determined with nanoDSF.Data analyzing was done using standard procedures with the software PRStability Analysis V.1.0.3. Ton is the onset temperature at whichproteins begin to unfold. The inflection points (IP) are the maxima fromthe first derivative of the melting curves. All data were measured induplicates, shown are mean data. Variant T_(on) [° C.] IP #1 [° C.] IP#2 [° C.] IP#3 [° C.] GLP1-Fc01 57.39 65.90 88.92 — GLP1-Fc02 61.9069.62 80.72 — GLP1-Fc03 47.20 60.26 78.95 — GLP1-Fc04 48.41 61.39 70.8487.87 GLP1-Fc05 55.86 65.36 84.74 — GLP1-Fc06 57.05 67.12 74.79 85.53GLP1-Fc07 53.47 64.86 85.40 — GLP1-Fc08 59.65 69.35 84.76 — GLP1-Fc0961.02 70.63 84.88 — GLP1-Fc10 58.88 69.43 84.60 — GLP1-Fc11 54.42 64.9485.04 — GLP1-Fc12 54.81 64.99 86.66 — GLP1-Fc13 59.34 69.00 87.01 —GLP1-Fc14 60.75 70.28 86.97 —

GLP1-Fc01 (the unmodified IgG4 backbone) shows one major unfoldingevent. GLP1-Fc02 (the unmodified IgG1 backbone) resulted in a morestabilized protein and a second unfolding event is clearly detectable.GLP1-Fc03 (the variant without any covalent bond between the heavychains) is more thermolabile than GLP1-Fc01 and GLP1-Fc02. GLP1-Fc04 (FcIgG4 variant according to the present invention) is slightly more stablethan GLP1-Fc03. In GLP1-Fc02 and GLP1-Fc03 the CH3 domain is derivedfrom IgG1 whereas in GLP-Fc01 and GLP1-Fc04 the CH3 domain is from IgG4.It is evident, that a clear second unfolding event is only measured invariants with an IgG1 derived CH3 domain (FIG. 12A). Based on thesedata, the IgG1 Fc backbone was chosen for further engineering. Aninternal disulfide-bond was introduced into the CH2 domain to furtherimprove stability in the Fc variants according to the present inventionGLP1-Fc06-GLP1-Fc10. While GLP1-Fc06 showed a slightly higherthermostability and GLP-Fc07 showed no stabilization at all, theintroduced disulfide bond in GLP1-Fc08, GLP1-Fc09, and GLP1-Fc10resulted in proteins with comparable stability as the IgG1 LALA Fcbackbone (GLP1-Fc-02; FIG. 12 , Tab. 6).

To further stabilize the Fc variants according to the present invention,the linker sequence between the Fc region and the C-terminal sequencecorresponding to the hinge region was omitted (GLP1-Fc11). In anotherapproach, the C-terminal sequence corresponding to the hinge region wasshortened to a proline-cysteine rich sequence (GLP1-Fc12). For theseproteins, an increase in thermostability for the second unfolding event(CH3 domain) was achieved (FIG. 12C, Tab. 6). The stabilizing effects ofan internal disulfide bond within the CH2 domain and a shortenedproline-cysteine rich sequence are combined in GLP1-Fc13 and GLP1-Fc14(FIG. 12D, Tab. 6).

Functionality In Vivo

In GLP1-Fc04 the original hinge was not deleted but the cysteines weremutated to serines. This was done to have an Fc variant of the GLP1-RAFc fusion protein according to the present invention which is as similaras possible to the parent molecule and this variant was chosen for ahead-to-head pharmacodynamic analysis. Both proteins were administereds.c. every fourth day in db/db-mice for 14 days and blood glucose aswell as body weight was monitored (FIG. 13 ). Mean body weights at day−1 (n=8 for each group) were 24.9 g (lean vehicle control), 54.2 g(db/db vehicle control), 55.4 g (GLP1-Fc01 group), and 54.8 g (GLP1-Fc04group). After the treatment period mean body weights were 25.2 g (leanvehicle control, +1.2%), 57.7 g (db/db vehicle control, +6.5%), 53.8 g(GLP1-Fc01 group, −2.9%), and 50.4 g (GLP1-Fc04 group, −2.6%). For thelean vehicle control body weight remained nearly constant over thetreatment period whereas the db/db vehicle control gained body weight.Both treated groups lost body weight at a comparable range. Bothcompounds, GLP1-Fc01 and GLP1-Fc04, show undistinguishable effects onthe change in blood glucose levels. These findings suggest that theGLP1-RA maintains its full activity when fused to the Fc variantaccording to the present invention. The coincident profile of bloodglucose change over the time suggest, that the recycling function of theFc variant according to the present invention is not compromised invivo.

Disclosed items

Disclosed is also the Following:

-   -   1. A molecule comprising two polypeptides,        -   wherein said polypeptides form an antibody Fc region,        -   wherein said two polypeptides are linked by at least two            covalent bonds which are located C-terminally to the portion            of each polypeptide which forms the Fc region,        -   wherein said two polypeptides are not linked by a disulfide            bond located N-terminally to the portion of each polypeptide            which forms the Fc region.    -   2. The molecule of claim 1, wherein the two covalent bonds are        two disulfide bonds.    -   3. The molecule of any of the preceding claims, wherein the at        least two covalent bonds located C-terminally to the portion of        each polypeptide which forms the Fc region are comprised in a        sequence corresponding to the amino acid sequence of an antibody        hinge region.    -   4. The molecule of any of the preceding claims, wherein the        affinity to C1q and/or FcγR1, FcγR2 and/or FcγR3 is reduced at        least 10-fold, compared to the same molecule comprising an        antibody hinge region located N-terminally to the portion of        each polypeptide which forms the Fc region.    -   5. The molecule of any of the preceding claims, wherein the        affinity to protein A and/or FcgRn is not reduced, compared to        the same molecule comprising an antibody hinge region located        N-terminally to the portion of each polypeptide which forms the        Fc region.    -   6. The molecule of any of the preceding claims, wherein the Fc        region is the Fc region of IgG, IgM, IgA, IgD or IgE.    -   7. The molecule of any of the preceding claims, wherein the at        least two covalent bonds are located C-terminally to a CH2, CH3        and/or CH4 domain on each polypeptide.    -   8. The molecule of any of the proceeding claims, wherein the Fc        region is the Fc region of an IgG antibody.    -   9. The molecule of any of the preceding claims, wherein the at        least two covalent bonds are located C-terminally to a CH2        and/or CH3 domain on each polypeptide.    -   10. The molecule of any of the preceding claims, wherein the at        least two covalent bonds are located C-terminally to a CH2 and        CH3 domain on each polypeptide.    -   11. The molecule of any of the preceding claims, wherein each        polypeptide comprises a CH2 domain which is part of the Fc        region, wherein each of these CH2 domains comprises an internal        disulfide bond which is not present in the corresponding        wild-type CH2 domain.    -   12. The molecule of claim 11, wherein the additional internal        disulfide bond is selected from the group consisting of        disulfide bonds formed between cysteine residues at the        following positions according to EU numbering: P238C and D265C;        P238C and A327C; P238C and L328C; S267C and A327C; and V240C and        I332C.    -   13. The molecule of any of the preceding claims, wherein each of        the polypeptides comprises a linker, wherein the linker links        the C-terminus of portion of the polypeptide forming the Fc        region to the at least two covalent bonds located C-terminally        to said portion of the polypeptide forming the Fc region.    -   14. The molecule of any of the preceding claims, wherein the        molecule further comprises at least one active moiety.    -   15. A polynucleotide encoding a molecule according to any of the        preceding claims.

1. A molecule comprising two polypeptides, wherein said polypeptidesform an antibody Fc region, wherein said two polypeptides are linked byat least two covalent bonds which are located C-terminally to theportion of each polypeptide which forms the Fc region, wherein said twopolypeptides are not linked by a disulfide bond located N-terminally tothe portion of each polypeptide which forms the Fc region, wherein eachpolypeptide comprises a CH2 domain which is part of the Fc region,wherein each of these CH2 domains comprises an additional internaldisulfide bond which is not present in the corresponding wild-type CH2domain, wherein the additional internal disulfide bond is selected fromthe group consisting of disulfide bonds formed between cysteine residuesat the following positions according to EU numbering: P238C and D265C;P238C and L328C; S267C and A327C; and V240C and I332C.
 2. The moleculeof claim 1, wherein the additional internal disulfide bond is selectedfrom the group consisting of disulfide bonds formed between cysteineresidues at the following positions according to EU numbering: P238C andL328C; S267C and A327C; and V240C and I332C.
 3. The molecule of claim 1,wherein the additional internal disulfide bond is selected from thegroup consisting of disulfide bonds formed between cysteine residues atthe following positions according to EU numbering: P238C and L328C; andS267C and A327C.
 4. The molecule of claim 1, wherein the two covalentbonds are two disulfide bonds.
 5. The molecule of claim 1, wherein theat least two covalent bonds located C-terminally to the portion of eachpolypeptide which forms the Fc region are comprised in a sequencecorresponding to the amino acid sequence of an antibody hinge region. 6.The molecule of claim 1, wherein the affinity to C1q and/or FcγR1, FcγR2and/or FcγR3 is reduced at least 10-fold, compared to the same moleculecomprising an antibody hinge region located N-terminally to the portionof each polypeptide which forms the Fc region.
 7. The molecule of claim1, wherein the affinity to protein A and/or FcgRn is not reduced,compared to the same molecule comprising an antibody hinge regionlocated N-terminally to the portion of each polypeptide which forms theFc region.
 8. The molecule of claim 1, wherein the Fc region is the Fcregion of IgG, IgM, IgA, IgD or IgE.
 9. The molecule of claim 1, whereinthe at least two covalent bonds are located C-terminally to a CH3 or CH4domain on each polypeptide.
 10. The molecule of claim 1, wherein the Fcregion is the Fc region of an IgG antibody.
 11. The molecule of claim 1,wherein the at least two covalent bonds are located C-terminally to aCH3 domain on each polypeptide.
 12. The molecule of claim 1, wherein themolecule further comprises at least one active moiety.
 13. The moleculeof claim 12, wherein the active moiety is located N-terminal of the CH2domain of at least one polypeptide and the molecule does not comprise anactive moiety which is located C-terminal of the at least two covalentbonds which are located C-terminally to the portion of each polypeptidewhich forms the Fc region.
 14. The molecule of claim 12, wherein theactive moiety is an antigen-binding moiety.
 15. A polynucleotideencoding a molecule according to claim 1.